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SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals.

Brickner JH, Fuller RS - J. Cell Biol. (1997)

Bottom Line: Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p.This hypothesis was confirmed in several ways.TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.

ABSTRACT
Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail). Mutation of TLS1 results in rapid transit of Kex2p to the vacuole. Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes. SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans. Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form. Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor. Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p. These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC). This hypothesis was confirmed in several ways. Soi1p was shown to be required for optimal function of TLS1. Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail. TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step. We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.

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TLS2 slows delivery of Kex2p to the PVC. (A) Mutation of TLS1 does not affect the rate of delivery to the PVC.  Strain 0472-28 (vps28 KEX2) expressing either I718tail Kex2p  (pCWKX10-I718tail; top panel) or Y713A I718tail Kex2p  (pCWKX11-I718tail; bottom panel) on a CEN plasmid was pulse-labeled for 10 min at 30°C in SDC-Met-Ura and chased for 80  min (see Materials and Methods). At the indicated times after addition of chase, cells were collected, lysed, and immunoprecipitated using antisera against the Kex2p lumenal domain. After  SDS PAGE, WT Kex2p, I718tail Kex2p, or Y713A I718tail Kex2p  were quantified, and t1/2 values were obtained by linear regression. Indicated are the positions of WT Kex2p (Full length; filled  arrows), I718tail Kex2p, or Y713A I718tail Kex2p (open arrows).  (B) Strain 0472-28 containing pCWKX10-I718tail was grown at  23°C in SD + Ade, His, Leu, Lys, and Trp, labeled, chased, and  processed for immunoprecipitation as described in A. Note that  under otherwise identical conditions, the rate of degradation of  Kex2p in the vps28 mutant is slower in SDC-Met-Ura than in SD  + Ade, His, Leu, Lys, and Trp (data not shown). This may be  caused by increased proteolytic activity in the PVC caused by  amino acid limitation.
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Figure 10: TLS2 slows delivery of Kex2p to the PVC. (A) Mutation of TLS1 does not affect the rate of delivery to the PVC. Strain 0472-28 (vps28 KEX2) expressing either I718tail Kex2p (pCWKX10-I718tail; top panel) or Y713A I718tail Kex2p (pCWKX11-I718tail; bottom panel) on a CEN plasmid was pulse-labeled for 10 min at 30°C in SDC-Met-Ura and chased for 80 min (see Materials and Methods). At the indicated times after addition of chase, cells were collected, lysed, and immunoprecipitated using antisera against the Kex2p lumenal domain. After SDS PAGE, WT Kex2p, I718tail Kex2p, or Y713A I718tail Kex2p were quantified, and t1/2 values were obtained by linear regression. Indicated are the positions of WT Kex2p (Full length; filled arrows), I718tail Kex2p, or Y713A I718tail Kex2p (open arrows). (B) Strain 0472-28 containing pCWKX10-I718tail was grown at 23°C in SD + Ade, His, Leu, Lys, and Trp, labeled, chased, and processed for immunoprecipitation as described in A. Note that under otherwise identical conditions, the rate of degradation of Kex2p in the vps28 mutant is slower in SDC-Met-Ura than in SD + Ade, His, Leu, Lys, and Trp (data not shown). This may be caused by increased proteolytic activity in the PVC caused by amino acid limitation.

Mentions: We analyzed the rate of turnover of WT Kex2p coexpressed with either I718tail Kex2p or Y713A I718tail Kex2p in a vps28 strain (Fig. 10). All three forms of Kex2p were degraded rapidly in the vps28 strain at 30°C (Fig. 10 A), with I718tail Kex2p and Y713A I718tail Kex2p exhibiting nearly identical t1/2 values of 24 and 23 min, respectively (Fig. 10). This indicated that TLS1 has no effect on the rate of delivery to the PVC and likely functions to promote retrieval to the TGN. There was only a small difference between the rates of delivery of I718tail Kex2p and of WT Kex2p (i.e., full-length; t1/2 = 27 min ± 1 min) to the PVC. To amplify the possible difference between WT Kex2p and I718tail Kex2p in this assay, we compared the t1/2 values of the two proteins in the vps28 mutant at 23°C (see Materials and Methods). Under these conditions, WT Kex2p was degraded with a t1/2 of 37 min ± 2 min, while I718tail Kex2p was degraded with a t1/2 of 27 min ± 3 min (n = 2; Fig. 10 B). Thus, TLS2 slows the rate of delivery of Kex2p to the PVC. Together with the observation that activation of TLS2 leads to an increase in the concentration of Y713A Kex2p in the TGN (Fig. 8 A), these data suggest that TLS2 regulates the rate of exit of Kex2p from the TGN.


SOI1 encodes a novel, conserved protein that promotes TGN-endosomal cycling of Kex2p and other membrane proteins by modulating the function of two TGN localization signals.

Brickner JH, Fuller RS - J. Cell Biol. (1997)

TLS2 slows delivery of Kex2p to the PVC. (A) Mutation of TLS1 does not affect the rate of delivery to the PVC.  Strain 0472-28 (vps28 KEX2) expressing either I718tail Kex2p  (pCWKX10-I718tail; top panel) or Y713A I718tail Kex2p  (pCWKX11-I718tail; bottom panel) on a CEN plasmid was pulse-labeled for 10 min at 30°C in SDC-Met-Ura and chased for 80  min (see Materials and Methods). At the indicated times after addition of chase, cells were collected, lysed, and immunoprecipitated using antisera against the Kex2p lumenal domain. After  SDS PAGE, WT Kex2p, I718tail Kex2p, or Y713A I718tail Kex2p  were quantified, and t1/2 values were obtained by linear regression. Indicated are the positions of WT Kex2p (Full length; filled  arrows), I718tail Kex2p, or Y713A I718tail Kex2p (open arrows).  (B) Strain 0472-28 containing pCWKX10-I718tail was grown at  23°C in SD + Ade, His, Leu, Lys, and Trp, labeled, chased, and  processed for immunoprecipitation as described in A. Note that  under otherwise identical conditions, the rate of degradation of  Kex2p in the vps28 mutant is slower in SDC-Met-Ura than in SD  + Ade, His, Leu, Lys, and Trp (data not shown). This may be  caused by increased proteolytic activity in the PVC caused by  amino acid limitation.
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Figure 10: TLS2 slows delivery of Kex2p to the PVC. (A) Mutation of TLS1 does not affect the rate of delivery to the PVC. Strain 0472-28 (vps28 KEX2) expressing either I718tail Kex2p (pCWKX10-I718tail; top panel) or Y713A I718tail Kex2p (pCWKX11-I718tail; bottom panel) on a CEN plasmid was pulse-labeled for 10 min at 30°C in SDC-Met-Ura and chased for 80 min (see Materials and Methods). At the indicated times after addition of chase, cells were collected, lysed, and immunoprecipitated using antisera against the Kex2p lumenal domain. After SDS PAGE, WT Kex2p, I718tail Kex2p, or Y713A I718tail Kex2p were quantified, and t1/2 values were obtained by linear regression. Indicated are the positions of WT Kex2p (Full length; filled arrows), I718tail Kex2p, or Y713A I718tail Kex2p (open arrows). (B) Strain 0472-28 containing pCWKX10-I718tail was grown at 23°C in SD + Ade, His, Leu, Lys, and Trp, labeled, chased, and processed for immunoprecipitation as described in A. Note that under otherwise identical conditions, the rate of degradation of Kex2p in the vps28 mutant is slower in SDC-Met-Ura than in SD + Ade, His, Leu, Lys, and Trp (data not shown). This may be caused by increased proteolytic activity in the PVC caused by amino acid limitation.
Mentions: We analyzed the rate of turnover of WT Kex2p coexpressed with either I718tail Kex2p or Y713A I718tail Kex2p in a vps28 strain (Fig. 10). All three forms of Kex2p were degraded rapidly in the vps28 strain at 30°C (Fig. 10 A), with I718tail Kex2p and Y713A I718tail Kex2p exhibiting nearly identical t1/2 values of 24 and 23 min, respectively (Fig. 10). This indicated that TLS1 has no effect on the rate of delivery to the PVC and likely functions to promote retrieval to the TGN. There was only a small difference between the rates of delivery of I718tail Kex2p and of WT Kex2p (i.e., full-length; t1/2 = 27 min ± 1 min) to the PVC. To amplify the possible difference between WT Kex2p and I718tail Kex2p in this assay, we compared the t1/2 values of the two proteins in the vps28 mutant at 23°C (see Materials and Methods). Under these conditions, WT Kex2p was degraded with a t1/2 of 37 min ± 2 min, while I718tail Kex2p was degraded with a t1/2 of 27 min ± 3 min (n = 2; Fig. 10 B). Thus, TLS2 slows the rate of delivery of Kex2p to the PVC. Together with the observation that activation of TLS2 leads to an increase in the concentration of Y713A Kex2p in the TGN (Fig. 8 A), these data suggest that TLS2 regulates the rate of exit of Kex2p from the TGN.

Bottom Line: Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p.This hypothesis was confirmed in several ways.TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606, USA.

ABSTRACT
Localization of yeast Kex2 protease to the TGN requires a signal (TLS1) in its cytosolic tail (C-tail). Mutation of TLS1 results in rapid transit of Kex2p to the vacuole. Isolation of suppressors of the Tyr713Ala mutation in TLS1 previously identified three SOI genes. SOI1, cloned by complementation of a sporulation defect, encodes a novel, hydrophilic 3,144-residue protein with homologues in Caenorhabditis elegans, Drosophila melanogaster, and humans. Epitope-tagged Soi1p existed in a detergent-insensitive, sedimentable form. Deletion of SOI1 impaired TGN localization of wild-type Kex2p and a fusion protein containing the C-tail of Ste13p, and also caused missorting of carboxypeptidase Y and accelerated vacuolar degradation of the Vps10p sorting receptor. Deletion of SOI1 improved retention of Tyr713Ala Kex2p in the pro-alpha-factor processing compartment but, unlike the original soi1 alleles, did not increase the half-life of Tyr713Ala Kex2p. These results suggested that Soi1p functions at two steps in the cycling of Kex2p and other proteins between the TGN and prevacuolar compartment (PVC). This hypothesis was confirmed in several ways. Soi1p was shown to be required for optimal function of TLS1. Suppression of the Tyr713Ala mutation by mutation of SOI1 was shown to be caused by activation of a second signal (TLS2) in the Kex2p C-tail. TLS2 delayed exit of Kex2p from the TGN, whereas TLS1 did not affect this step. We propose that Soi1p promotes cycling of TGN membrane proteins between the TGN and PVC by antagonizing a TGN retention signal (TLS2) and facilitating the function of a retrieval signal (TLS1) that acts at the PVC.

Show MeSH
Related in: MedlinePlus